The oral cavity is inhabited by more than 500 species of Gram-positive and Gram-negative bacteria, which exist in a complex ecosystem referred to as a biofilm. Biofilm formation is initiated by the attachment of oral streptococci to the acquired salivary pellicle coating the surfaces of the oral cavity. As the biofilm develops and matures, conditions become propitious for the attachment of the late bacterial colonizers, which are associated with periodontal disease to the existing biofilm. Bacterial adhesion to the acquired salivary pellicle or to host receptors is a crucial step for biofilm formation, bacterial colonization and infection. Bacterial adhesion is mediated by fimbrial and non-fimbrial adhesins, which are proteinaceous appendages displayed on the surface of bacteria containing the receptor binding domains. Fimbrial adhesins form long filamentous structures composed of multiple copies of either a single subunit or multiple subunits. The long-fimbriae of S. parasanguis are composed of multiple subunits of Fap1 (fimbriae-associated protein 1), which are crucial for biofilm formation. Fap-1 homologues have recently been found in other streptococcal family members. Non-fimbrial adhesins form shorter structures comprised of either a single subunit or a small number of identical subunits specifically folding to expose the receptor binding domain at the distal-end. A. actinomycetemcomitans uses specific non-fimbrial adhesins to adhere to and invade into epithelial cells as well as interacting with extracellular matrix proteins. EmaA (Extracellular matrix protein adhesin-A) is a newly discovered non-fimbrial adhesin of A. actinomycetemcomitans involved in binding to collagen and tubulin in the host tissue. EmaA is a multimeric autotransporter protein, which forms structures on the bacterial cell surface by oligomerization of either 3 or 4 subunits (total molecular mass of 600-800 kDa). Structural information on this adhesin and/or the adhesin/receptor complexes is unknown. Detailed 3D structural information on adhesin/receptor interactions is extremely important for the development of next generation therapeutics due to the increase number of antibiotic resistant organisms. To date, however, there is a paucity of structural information regarding oral bacteria. In this application, we propose to determine the 3D structure of the adhesins and adhesin/receptor complexes of both an early bacterial colonizer (Fap-1) and a late colonizer (EmaA) essential to gain insights into the different stages of the pathogenic process. We will carry out these studies using novel techniques of electron microscopy and image processing. Our work will provide the first 3D structural data for these adhesins and the respective adhesin/receptor complexes. PROJECT NARRATIVE: The human oral cavity is inhabited by more than 500 species of bacteria, which are responsible for causing oral and serious extra-oral systemic diseases. Bacterial adhesion, mediated by adhesins on the cell surface, is a crucial step for biofilm formation, colonization, and infection. We will determine the 3D structure of the adhesins and adhesin/receptor complexes, which is essential to gain insights into the different stages of the pathogenic process.